Chromatin structure and architecture. We are interested in the biophysical and structural properties of native chromatin fragments. Using the extensively studied chicken folate receptor and beta-globin gene loci, we have previously characterized the hydrodynamic and gross structural properties of two distinct chromatin fragments. The one fragment represents a constitutively condensed heterochromatin region spanning 15.5 Kbp of DNA flanked by the developmentally regulated folate receptor and beta-globin genes. The second fragment, which is released from the transcriptionally inactive beta-globin gene locus, spans 16.2 Kbp of DNA. Despite their different histone protein to nucleic acid ratio, we have shown that both fragments adopt extended rod like structures consistent with models proposed for the condensed 30 nm chromatin fiber. A variety of models have been proposed to describe the condensed 30 nm chromatin fiber, each having a topologically distinct DNA path for essentially the same arrangement of nucleosomes. In order to characterize the spatial arrangement of the DNA within chromatin we are currently developing high resolution chromosome capture conformation assays utilizing both in vitro model systems, as well as native chromatin fragments. These studies will allow us to assign the appropriate 30 nm fiber model to chromatin, which will in turn provide a better understanding of the relations between chromatin structure and essential processes such as gene expression and DNA replication. Macromolecular assemblies. In collaboration with members of the Laboratory of Molecular Biology, and others, protein and protein-nucleic acid assemblies have been characterized in terms of their shape, stoichiometry and affinity of interaction using hydrodynamic methods. These studies extend current biochemical and structural investigations and provide complementary mechanistic information as exemplified by recently published studies on Enzyme I (EI) carried out in collaboration with Dr. G. Marius Clore. The phosphoenolpyruvate:sugar phosphotransferase system is a bacterial signal transduction pathway in which active sugar transport across the cell membrane is coupled to a sequential phosphorylation cascade. The initial two steps are common to all branches of the pathway and involve the autophosphorylation of EI by phosphoenolpyruvate (PEP), followed by phosphoryl transfer from EI to the histidine phosphocarrier protein HPr. The phosphoryl group is subsequently transferred from HPr to the sugar specific enzyme II, and ultimately onto the incoming sugar molecule. EI consists of an N-terminal phosphoryl transfer domain (EIN) that binds HPr and a C-terminal dimerization domain (EIC) that contains the PEP binding site. We have characterized the monomer-dimer equilibria of EI under various conditions and determined an affinity of 0.8 micromolar in the presence of 100 mM sodium chloride and 4 mM magnesium chloride. These conditions were therefore chosen for solution structural studies as the protein will be predominantly dimeric at the millimolar concentrations used. These structural studies show that that the EIN domains of undergo large hinge body rotations when bound by HPr, thus providing important mechanistic information on the catalytic cycle of the dimeric EI. Furthermore, the structural data are consistent with hydrodynamic parameters determined during the characterization of the EI self-association (Schwieters et al., 2010).